Liposome vesicle precursors

ABSTRACT

The invention relates to a dry deposit as a precursor to liposome vesicles, the precursor being a three dimensional expanded structure with bulk density between 0.01 and 0.001 g/cm 3 . The invention also concerns a method of making liposome vesicles with an enhanced entrapment capacity by dissolving one or more film forming lipids in at least one organic solvent to form a solution in a reaction vessel, evaporating the solvent to form an expanded three dimensional porous lipid structure, contacting the lipid deposit with an aqueous carrier phase, and producing liposome vesicles entrapping the carrier phase as well as an apparatus comprising an array of tubing or an inert packing which serves as a material support or a matrix surface for the deposition of lipids produced according to the method.

This is a rule 60 division of application Ser. No. 08/527,087, filedSep. 12, 1995 now U.S. Pat. No. 5,702,722.

TECHNICAL FIELD

The invention relates to a liposome vesicle precursor in the form of adry lipid deposit and a method of making liposome vesicles with enhancedentrapment capacity by dissolving one or more film forming lipids in atleast one organic solvent in a reaction vessel, depositing the lipids byevaporation of the solvent, contacting the lipid deposit with an aqueoussolution carrier phase, and producing liposome vesicles entrapping thesolution. The invention also concerns a an apparatus for carrying outthe method, contrast agents comprising the liposome vesicle precursorand a method of making contrast agents using the precursor.

BACKGROUND ART

Liposomes vesicles whose binding envelope consists of bi- or multilayerof lipid molecules have been long recognised as drug delivery systemswhich can improve therapeutic and diagnostic effectiveness of many drugsand contrast agents. Experiments with a number of different antibioticsand X-ray contrast agents have shown that better therapeutic activity orbetter contrast with a higher level of safety may be achieved byencapsulating drugs and contrast agents with liposomes. Great interestin liposomes as encapsulating systems for drugs has revealed that asuccessful development and commercialisation of such products requiresreproducible methods of large scale production of lipid vesicles withsuitable characteristics. Consequently, a search for methods which willconsistently produce liposome vesicles of the required size andconcentration, size distribution and entrapping capacity regardless ofthe nature of lipid mixture have been initiated. Such methods ought toprovide liposomes with consistent active substance to lipid ratio whilerespecting currently accepted good manufacturing practices. As a resultof the search, and due to the fact that the liposome behaviour can varysubstantially with various production parameters, many different methodsof manufacture have been proposed so far.

Conventional liposome preparation methods include a number of steps inwhich multi- or the bilayer-forming components (phospholipids ormixtures of phospholipids with other lipids e.g. cholesterol) aredissolved in a volatile organic solvent or solvent mixture in a roundbottom flask followed by evaporation of the solvent under conditions(temperature and pressure) which will prevent phase separation. Uponsolvent removal, a dry lipid mixture, usually in form of a film depositon the walls of the reactor, is hydrated with an aqueous medium whichmay contain dissolved buffers, salts, conditioning agents and an activesubstance to be entrapped. Liposomes will form in the hydration stepwhereby a proportion of the aqueous medium becomes encapsulated in theliposomes. The hydration can be performed with or without energising thesolution by means of stirring, sonication or microfluidisation withsubsequent extrusion through one or more polycarbonate filters. The freenon-encapsulated active substance can be separated for recovery and theproduct is filtered, sterilised, optionally lyophilised, and packed.

Hydration, more than any other step, influences the type of liposomesformed (size, number of lipid layers, entrapped volume). The nature ofthe dried lipid, its surface area, and its porosity are of particularimportance. Thus it has been established that the hydration andentrapping process are most efficient when the film of dry lipids iskept thin. This means that greater the lipid quantity, greater thesurface for deposition of the lipids is required, it also means thateven though glass beads and other inert insoluble particles are used toincrease the surface area available for film deposition, the thin filmmethod remains largely a laboratory method.

Other methods of making liposomes involving injection of an organicsolutions of lipids into an aqueous medium with continuous removal ofsolvent, use of spray drying, lyophilization, microemulsification andmicrofluidization, etc. have been proposed a number of publications orpatents such as for example U.S. Pat. No. 4,529,561, U.S. Pat. No.4,572,425, etc.

An attempt to solve problems of the scale-up of liposome production hasbeen described in the U.S. Pat. No. 4,935,171 (Vestar). There isdisclosed a method for preparing liposomes in commercial quantities byforming a homogeneous and uniform lipid film in a thin film evaporatorthrough evaporation of the organic solvent. After drying of the thinlipid film which is formed on the inner wall of the evaporator, thedeposit is in situ hydrated with an aqueous phase under agitationprovided by the rotor. Although the solution proposed in this documentseems to be a step in the right direction the lipid film surface to thereactor volume ratio is only slightly, if not marginally, better thanthat of the round-bottom flasks used on laboratory scale. The reactor'sspace time yield or productivity is still far too low for the process tobe economically sound and competitive.

Different aspects of the liposome manufacturing have been addressed anda number of improvements and different solutions to the problem ofscale-up have been proposed. Documents such as for exampleWO-A-86/00238, WO-A-87/00043, U.S. Pat. No. 4,737,323, U.S. Pat. No.4,753,788, and U.S. Pat. No. 4,781,871 have suggested use of rapidfreezing of previously prepared multi lamellar vesicles with subsequentfreeze and thaw treatment to improve their entrapment capacity, use ofextrusion technique of multilamellar liposomes to improve their sizedistribution, etc.

So far there has been no suggestion towards a large scale industrialmethod whose control of production parameters will allow reproducibleprocess in which large volumes of liquid will be processed within arelatively small reactor space. All known processes of pilot orindustrial scale would, typically, be linked to small batches in whichprocessing of large volumes of dilute liposome solutions would require alot of floor and reactor space as well as handling of large volumes ofsolutions and solvents. In reality due to relatively low space timeyields or productivity of reactors these methods are too cumbersome andfar too costly for a large scale commercial production.

SUMMARY OF THE INVENTION

Briefly summarised the invention relates to a supported or unsupportedliposome vesicle precursor in the from of a three dimensional structureof expanded lipids with bulk density below 0.1 g/cm³ preferably below0.08, more preferably between 0.05 and 0.001 and even more preferablybetween 0.02 and 0.01. By supported structure it is meant that the lipidporous deposit is formed on an array or network of inert supportingmaterial. The lipids forming the deposit are selected from synthetic ornatural, saturated and unsaturated phospholipids including phosphatidicacid, phosphatidyl choline, phosphatidylethanol amine, phosphatidylserine, phosphatidyl glycerol, phosphatidyl inositol and mixturesthereof. The lipids may further contain substances selected fromdicetylphosphate, cholesterol, ergosterol, phytosterol, sitosterol,lanosterol, α-tocopherol, stearic acid, stearyl amine and mixturesthereof.

The invention also concerns a method of making liposome vesicles withenhanced entrapment capacity by dissolving one or more film forminglipids in at least one organic solvent to form a solution. The solutionof lipids is introduced into a suitable reaction vessel and subjected toevaporation whereby the drying lipids form expanded three dimensionalporous structure whose bulk density is below 0.1 g/cm³, preferably below0.08, more preferably between 0.05 and 0.001 and even more preferablybetween 0.02 and 0.01. Thereafter, the porous structure is contactedwith an aqueous carrier phase to produce liposome vesicles entrapping aportion of the carrier phase.

The invention further comprises an apparatus for the manufacture ofliposomes with high entrapment capacity according to the above methodcomprising a reaction vessel with an inlet and an outlet, a connectionto a vacuum, means for cooling or heating, a control means, and apacking comprising an array of a closely packed tubing of an inertmaterial. Preferably, the tubing is stainless steel tubing with theinner diameter of between 0.5 mm and 5 mm and the wall thickness ofbetween 0.5 mm and 2 mm. Alternatively, the packing, which may bestationary or moving e.g. fluidized, may comprise Raschig rings, hollowglass spheres, reticulated carbon, reticulated vitreous carbon,reticulated metal, glass or metal wool and glass or metal fibre.

The three dimensional lipid structures of the invention are verysuitable for a large scale manufacture of liposomes with high entrapmentcapacity.

When incubated with an aqueous carrier phase containing a contrastmedium, the three dimensional lipid structures of the invention areparticularly suitable for the manufacture of diagnostic contrast agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B are a schematic diagram of the reactor with a cut-outshowing the expanded three dimensional lipid structure of the invention.

FIG. 2 is a schematic diagram of the reactor with an array of an inerttubing.

FIG. 3 is a plot of lipid deposition vs concentration.

FIG. 4 is a plot of lipid deposition vs linear velocity.

FIG. 5 is a flow chart of the production of a contrast medium using theexpanded lipid structures of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the unexpected finding that optimal liposomeformation and enhanced reactor capacity are obtained if during theproduction of the vesicles, the lipid deposit obtained by evaporation ofthe solvent from an organic solution of one or more film forming lipidsin at least one organic solvent, prior to contacting with an aqueouscarrier phase, is expanded into a three dimensional structure whose bulkdensity is below 0.1 g/cm³, preferably below 0.08, more preferablybetween 0.05 and 0.001 and even more preferably between 0.02 and 0.01.Although the exact reasons for such unexpected results have not beenthoroughly established, it is assumed that the method provides anexceptionally large surface to volume ratio of the deposit whosesubsequent hydration is therefore more efficient. High yields ofliposomes of the desired size and distribution are thus produced by themethod which is particularly easy to scale up and control. Having alarge surface to volume ratio the expanded lipid structures improve thespace time yield of reactors, whereby the technique becomes industriallyvery attractive. In addition, to ease scale-up and promote highproductivity, the method provides further advantages which includefaster reactor turn around time, ease of control of the hydration step,reduced processing times, use of inexpensive materials, and finally useof the same reactor for deposition, solvent evaporation, hydration ofthe expanded lipid structure and sterilization of the liposome vesiclesformed.

It has been established that the porous structures of pure lipids of theinvention have very large surface to volume ratio. Unfortunately, due tothe great fragility of the expanded structure the exact surface area ofthe unit of volume or weight of the expanded lipid structure could nothave been established with great accuracy. However, a conservativeestimate of the total surface area of 1 g of the expanded structure ofthe invention suggests that the total surface area may vary between 0.1and 50 m² which implies surface to volume ratios of between 10 to0.5×10⁵.

The expanded three dimensional lipid structure may be obtained throughevaporation of the organic solvent from a reaction vessel which containsan inert porous network or a support which serves as a material supportor a matrix surface for the deposition of lipids. The inert network maybe any convenient material with a relatively large surface to volumeratio and it may include an array of tubing or an array of inert packingsuch as hollow glass spheres, reticulated carbon, reticulated vitreouscarbon, reticulated metal, glass, ceramic or metal wool and glass,ceramic or metal fibre. When an array of tubing is used the tubingdimensions should be chosen such that maximal ratio of surface to volumeis achieved. The experiments carried out in the course of thedevelopment and characterisation of the reactor according to theinvention have shown that in a given configuration the tubing with aninner diameter of between 0.5 mm and 5 mm and wall thickness between 0.5and 2 mm has provided favourable results, however, another configurationof the reactor may favour other tubing dimensions. It has beenestablished that since the lipid solution is spread over the inner andouter surface of the tubing by gravity an array of vertically arrangedtubing is preferred although a helical arrangement is also possible.

In order to facilitate uniform deposition of the lipid films the inertpacking may be gently fluidized or the reactor packed with Raschig ringsor any other inert material such as that mentioned above may be fed withthe lipid solution from the top and left to gently trickle down thepacking. It is believed that excellent results obtained in the trickletower arrangement come from the fact that an efficient control ofdeposit thickness is achieved by the trickle fashion of contacting ofthe lipid solution and the support. The excess liquid being constantlyremoved whereby uniform liquid thickness on the whole surface of thesupport is ensured. To further assist uniform formation of the coatingof the lipid solution on the packing air or an inert gas such asnitrogen may be introduced in counter-current fashion for a period oftime. The gas is usually cold however, under certain conditions it maybe desirable that the temperature of the gas is chosen such that dryingand expansion of the lipid film is assisted or performed using a hotgas.

After drying and expansion of the deposit consisting of pure or lipidswith usual degree of purity into the three dimensional structure, thedeposit is contacted with an aqueous carrier phase. Depending on theconfiguration of the reactor the carrier phase may be introduced at thelower end of the reactor e.g. in the case of trickle tower or fluidizedbed configuration or at the top of the reactor column (in case of thefixed array of tubing). The aqueous carrier phase used may be pure or itmay contain biologically active substances, contrast agents or both.Virtually any biologically active substance can be entrapped in theliposomes produced according to the invention. Such substances includebut are not limited to antibacterial compounds such as gentamycin,antiviral compounds such as rifamycins, antifungal compounds such asamphotericin B, antiparasitic compounds such as derivatives of antimony,antineoplastic compounds such as mitomycin C, doxorubicin andcisplatinum, proteins such as albumin and lipo-proteins,immunoglobulines, toxins such as diphteria toxin, enzymes such ascatalase, hormones, neurotransmitters, radio-opaque compounds such as ⁹⁹Tc, fluorescent compounds such as carboxy fluoroscein,anti-inflammatories such as salicylic acid and ibuprofen, anestheticssuch as dibucaine or lidocaine, etc.

Very good results and high entrapment loadings are achieved withiodinated X-ray contrast agents such as iopamidol, iomeprol, iohexol,iopentol, iotrolan, iodixanol, ioglucol, etc. The iodine to lipid ratioof the liposome vesicles according to the invention is at least 2.75.

The evaporation of the organic solvent or the mixure of solvents iscarried out at above ambient temperatures or reduced pressure or both.Experiments have shown that the rate of evaporation has a stronginfluence on the degree of expansion of the lipid structure. Hence foroptimal expansion, one will appropriately control the amount of heat andthe pressure within the reactor. The control becomes particularlyimportant near the end of solvent evaporation, i.e. when the solutionthickens and becomes viscous. At this point, a slight reduction ofpressure will result in a relatively fast expansion (foaming). It hasbeen established that by balancing the temperature and pressure for agiven solvent or solvent mixture different degrees of expansion of thelipid deposit may be achieved. Best results are obtained when theorganic solvent is selected from petroleum ether, chloroform, methanol,ethanol, propanol, isopropanol, n-butanol, tert-butanol, pentanol,hexanol, pentane, hexane, heptane, cyclohexane and mixtures thereof.Preferably the solvent is an azeotropic mixture of two solvents. Goodresults have been obtained with azeotropic mixtures of ethanol withcyclohexane, chloroform with methanol and iso-propanol with hexane.

Lipids used for production of liposome vesicles are conventional and areselected from synthetic or natural, saturated and/or unsaturatedphospholipids including phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol andphosphatidic acid. The following phospholipids are particularly usefuldipalmitoylphosphatidyl choline, dipalmitoylphosphatidyl glycerol,dipalmitoylphosphatidyl acid, dipalmitoylphosphatidyl ethanolamine andthe corresponding distearoyl- and dimyristyl- counterparts and mixturesthereof. Those lipids or their mixtures may further contain substancesselected from dicetylphosphate, cholesterol, ergosterol, phytosterol,sitosterol, lanosterol, α-tocopherol, stearic acid, stearyl amine andmixtures thereof.

The invention also includes a supported or unsupported three dimensionalstructure of expanded dry lipids with density of below 0.1 g/cm³,preferably below 0.08 g/cm³ and more preferably with the density ofbetween 0.05 and 0.01 g/cm³. By supported structure it is meant that thelipid porous deposit is formed on an array of inert supporting material.

The expanded three dimensional lipid structures are extremely useful forthe manufacture of liposomes with high entrapment capacity particularlywhen these liposomes are used to carry drugs or diagnostic contrastagents. In such a case the porous three dimenssional lipid structure iscontacted with an aqueous solution containing the drug or the diagnosticagent as an active ingredient whereby liposomes will form andencapsulate the ingredient. The liposomes carrying the active substanceare then processed as appropriate in a conventional way. Alternatively,a suspension of "empty" liposome vesicles i.e. liposome vesiclescontaining only aqueous liquid carrier may be formed first. In thesubsequent step these "empty" liposomes are contacted with a solutioncontaining an active ingredient and the vesicles loaded using forexample trans-membrane loading technique.

The invention further comprises an apparatus for the manufacture ofliposomes with high entrapment capacity comprising a reaction vesselwith an inlet and an outlet, a connection to a vacuum, means for coolingor heating, a control means, and a packing, characterised in that thepacking is an array of a closely packed tubing of an inert material. Thetubing having the inner diameter of between 0.5 mm and 5 mm and the wallthickness of between 0.5 and 2 mm is preferably arranged in a verticalfashion although a coil-like arrangement is also possible.

The following examples further illustrate the invention:

EXAMPLE 1

Reactor Characterisation

A vertical thermoregulated, 1 meter high, 316L stainless steel columnwith inner diameter of 50 mm fitted at its bottom end with a metal gridwas filled with 12 stainless steel tubes. The inner diameter of thetubing was 4 mm and wall thickness of 1 mm. Prior to insertion into thereactor the tubing was spot welded to form an array of parallel tubes.The same reactor configuration but with tubing of 2 mm and 3 mm diameterhave also been prepared and tested.

Prior to the tests directed to expansion of the lipid depositscharacterisation of the reactor was carried out by deposition ofnonexpanded lipid films using the following lipid composition:hydrogenated soy lecithin/dicetylphosphate in 9:1 molar ratio.Experiments were performed to determine the best conditions for thedeposition of the lipids in the tubes and to establish the impact of thelipid concentration, internal diameter and nature of the tubes and rateof drainage of the lipid solution. In all cases, the lipid solutions inchloroform were introduced into the tubes at room temperature and afterfilling of the tubes from the bottom the lipid solutions were drained ata controlled rate. The deposit was dried at 80° C. under nitrogen byevaporation of the solvent and the dry deposit rinsed 3 times with asmall amount of chloroform.

                  TABLE 1    ______________________________________                   Lipids deposited in    Lipid conc.    mg of lipid/100 cm.sup.2    g/l            3 mm tube                            4 mm tube    ______________________________________    180            33.1     23.3    220            43.7     29.7    260            62.5     40.0    300            81.5     56.5    320            94.5     80.4    340            111.3    84.2    ______________________________________

As shown in Table 1 and FIG. 3 the amounts of lipids deposited atvarious lipid concentrations and two different diameters of stainlesssteel tubing the lipid coating increases with an increase of the lipidconcentration. In addition, thicker deposits per unit area are obtainedin the 3 mm tubing than in 4 mm. In both cases the amounts of lipidsdeposited appear to be proportional to the square of the lipidconcentration.

As it can be seen from Table 2, the amount of lipids deposited increaseswith the rate of drainage. However, if these results are expressed as afunction of the drainage velocity (in cm per min) rather than drainageflow rate (in ml per min) amounts of the lipids deposited appear to benearly proportional to the linear velocity. That independently of theactual tube size. See FIG. 4.

                  TABLE 2    ______________________________________               Lipids deposited in mg lipid/100 cm.sup.2    Drainage rate               in a tube of    ml/min     2 mm        3 mm      4 mm    ______________________________________    1          55.2        55.3      37.4    2          76.5        63.0      47.3    3.75       114.9       83.7      65.4    5          121.0       94.5      80.4    ______________________________________

Additional experiments were carried out in the identical set up butusing glass tubed of stainless steel ones have shown that there are nomajor difference between the two supports with regard the lipiddesposition. The homogenity of the lipid coating was determined bycutting the coated steel tubes from the top in 10 cm intervals andmeasuring the film thickness. The results obtained are given in Table 3.

                  TABLE 3    ______________________________________                Lipids deposited in mg    Fraction      3 mm tube 4 mm tube    ______________________________________    1             5.15      5.13    2             6.63      3.77    3             7.22      4.52    4             8.20      3.50    5             7.59      5.58    6             6.55      6.16    7             5.59      6.38    8             5.63      6.21    9             5.22      6.97    10            9.06      6.86    Mean ± S.D.                  6.68 ± 1.33                            5.51 ± 1.24    ______________________________________

Calculation of apparent (bulk) densities of the lipid deposits obtainedin the three different reactor configurations have shown that for 2 mmtubing bulk densities were between 0.04 and 0.06 g/cm³, for 3 mm tubingbetween 0.02 and 0.04 g/cm³ and for 4 mm tubing the bulk densities werebetween 0.01 and 0.03 g/cm³.

Liposome production

After the characterisation, a new reactor with 250 stainless steel tubeswas made and connected into the circuit shown in FIG. 5. 26 g ofhydrogenated soy phosphatidylcholine (Nattermann) with 2 g ofdicetylphosphate and 106 g of chloroform were placed into 1 liter glassreactor equiped with stirrer, heating jacket, and condenser (1) andheated to 60° C. under stirring until complete dissolution. The lipidsolution filtered through the sterile filter (3) and loaded into 316 Lstainless steel column with a heating jacket filled with 250 of parallel1 m long 304 stainless steel tubes (4) by means of the peristaltic pump(2). The excess of solution was removed, the solvent evaporated and thelipids deposited at 80° C. by circulating air from the bottom of thecolumn.

The 2 liter glass reactor with stirrer, heating jacket, condenser (5)was filled with 849 g of iopamidol, 1196 g of water, 0.54 g of EDTA and1.60 g of Tris and heated at 90° C. under stirring until completesolubilization was obtained. The iopamidol solution was then filtered,transferred to the glass reactor (6) and therefrom to the column (4).The solution was circulated at 75° C. between the reactor (6) and thecolumn (4) by means of the gear pump (7). The liposome suspension formedwas then extruded through the filter (8), recovered in the reactor (9)and then concentrated using the microfiltration system (10) by means ofpump (11). The concentrated solution was washed with saline (12) toeliminate free iopamidol (diafiltration). Typical iodine to lipid ratios(I/L) for a number of experiments run under different experimentalcondition were in the range 2.5-3.5 with lipid concentrations between 25and 35 mg/ml with the liposome mean size of 570 nm.

The production unit can be sterilised (e.g. steam) and is envisaged as aclosed-circuit aseptic large scale production unit.

EXAMPLE 2

The Example 1 was repeated in the experimental set-up shown in FIG. 5but size was scaled-up by a factor four 518.6 g of hydrogenated soyphosphotidylcholine (Nattermann) with 41.4 g of dicetylphosphate andchloroform 2130.0 g were placed into 3 liter glass reactor equiped withstirrer, heating jacket, and condenser (1) and heated to 60° C. understirring until complete dissolution. The lipid solution was filtered onthe 0.22 μm sterile filter (3) using the peristaltic pump (2). The lipidsolution is then transfered into the 316 L stainless steel column with aheating jacket filled with 1000 of parallel 1 m long 304 stainless steeltubes (4) and the excess of the lipid solution removed. The chloroformwas evaporated and the lipids dry deposited at 80° C. by circulating airfrom the bottom of the column.

The 7 liter stainless steel (316L) reactor with stirrer, heating jacket,condenser (5) was loaded with 2920 g of iopamidol, 4110 g of water, 1.87g of EDTA and 5.50 g of Tris (HCl qsp for pH 7.2) and heated at 90° C.under stirring until complete solubilization was obtained. The iopamidolsolution was then passed through the sterile filter (not shown),transferred to column (4) using the gear pump (7) and circulated at 75°C. between the reactor (6) and the column (4) for a while. The liposomesuspension formed was recovered in the reactor (6), extruded through thefilter (8) at 75° C. and the liposomes recovered in reactor (9). Theliposome solution was then concentrated using the microfiltration system(10). Typical iodine to lipid ratios (I/L) for a number of experimentsrun under different experimental condition were in the range 2.5-3.5with lipid concentrations between 25 and 35 mg/ml with the liposome meansize of 570 nm. Bulk density of the lipid deposit varied as a functionof the experimental conditions and was estimated to be between 0.08 and0.05 g/cm³. However the best liposomes were prepared with the bulkdensities between 0.01 and 0.02 g/cm³.

EXAMPLE 3

The Example 2 was repeated using as solvent the azeotropic mixture ofchloroform and methanol (87/13=v/v). After evaporation of the solventunder reduced pressure 60° C. warm distilled water was added to thereactor. The temperature of the water added was above the transitiontemperature (54° C.) of the lipids used. The expanded three dimensionallipids deposit obtained was allowed to hydrate and the liposomes formedwere distributed homogeneously through the liquid. Liposomes of the MLVtype were formed in high yield. After about 1 hour, the liposomesuspension containing 5 mg/ml of lipids was extruded at 60° C. through a2 μm polycarbonate membrane (Nuclepore) and, after cooling to roomtemperature, it was concentrated to 30 mg/ml by microfiltration using a0.22 μm microfilter system Prostak (Millipore).

To the concentrated liposome suspension, there was added 1 liter of anaqueous solution containing 1040 g of (S)-N,N'-bis2-hydroxy-1-(hydroxymethyl)-ethyl!-2,4,6-triiodo-5-lactamido-isophtalamide(iopamidol) i.e. 520 g/l of covalent iodine at 60° C. The resultingmixture (2 l) with iodine concentration of 260 g/l was incubated forabout 30 min at 60° C., after which time the iodine concentrationoutside and inside the liposome core had equalized. The resultingpreparation was concentrated to 30 g lipids/l. The entrapped iodine tolipid ratio (I/L) was about 4.0.

EXAMPLE 4

A glass column (500 mm high and 50 mm in diameter) was filled withRaschig rings and operated as a trickle tower reactor. A lipid solutioncontaining 50 g/l of a mixture of distearoylphosphatidyl choline (DSPC),cholesterol and dicetylphosphate with molar ratio 5:4:1 in chloroformwas trickled down the column fitted with 35 layers of Raschig ringsspread over a nickel mesh as a support until the last layer at thebottom was thouroughly soaked with the solution. The excess of thesolution was removed and a stream of hot (80° C.) nitrogen blown fromthe bottom up through the reactor. The lipid deposit was dried for 2hours. The nitrogen flow stopped and the reactor connected to a vacuum(1-2 Torr) and the deposit allowed to dry until all chloroform wasremoved. After evaporation of the solvent iomeprol solution with iodineconcentration of 260 g/l was added at 60° C. to the reactor. Theexpanded three dimensional lipid deposit was allowed to hydrate for 30minutes. The liposomes suspension was extuded at 60° C. through a 2 μmpolycarbonate membrane (Nuclepore) and after cooling to room temperatureit was concentrated to 30 g lipids/l. The entrapped iodine to lipidratio (I/L) was above 4.0.

The same experiment was then repeated with reticulated carbon,reticulated nickel and reticulated glassy carbon as the column packing.Bulk densities of the three dimensional lipid stucture obtained in theseexperiments were between 0.05 and 0.005 g/cm³. Liposomes with lipid toiodine ratio of 3.5-4.5 were obtained.

EXAMPLE 5

A glass column (500 mm high and 50 mm in diameter) filled with hollowglass beads as an array of inert packing and operated as a fluidized bedreactor. A lipid solution containing 50 g/l of a mixture ofdipalmitoylphosphatidylcholine (DPPC), cholesterol anddipalmitoylphosphatidic acid (DPPA) with molar ratio 5:4:1 incyclohexane/ethanol azeotropic mixture (69.5/30.5 v/v) was introducedinto the column containing a 100 mm high bed of hollow glass spheressupported by a porous glass frit. The solution was allowed to thoroughlywet the glass beads and the excess removed. A stream of hot (80° C.) airwas blown from the bottom through the reactor and the spheres werefluidized until the lipid deposit was almost dry. The air flow was thenstopped, the reactor connected to a vacuum (1-2 Torr) and the depositallowed to dry until complete removal of solvents. After evaporation ofthe solvent, a 4% by weight lidocaine HCl solution in water (pH 7.2) at60° C. was added to the reactor. The liposome solution formed wasextruded at 80° C. through a 2 μm polycarbonate membrane (Nuclepore) andafter cooling to room temperature concentrated to 35 mg lipid/ml. Theentrapped lidocaine in liposomes was 0.35 mmol lidocaine/g lipid.

Various expansions (20-80%) of the bed during the fluidization showedlittle influence on the quality of the deposit.

EXAMPLE 6

The Example 4 was repeated using a 500 mm high glass column withoutinert packing. The column was filled with 100 ml of lipid solution (80g/l) prepared from azeotropic mixtures of ethanol/cyclohexane,chloroform/methanol and iso-propanol/hexane. The organic solvent wasfirst evaporated at 55° C. and 300 mmHg of pressure and then 70° C. and10 mmHg until formation of a foamed dry deposit. In all cases the threedimensional expanded lipid stucture obtained was then hydrated at 70° C.with an aqueous solution of iomeprol to produce liposome encapsulatediomeprol suspensions. After extrusion at 70° C. on a 0.6 μmpolycarbonate membrane (Nuclepore) the liposome suspension wasconcentrated. Typical iodine to lipid ratios (I/L) for a number ofexperiments run under different experimental conditions were in therange 1.9-2.5 with lipid concentrations between 25 and 35 mg/ml. Bulkdensities of the expanded lipid structure were estimated to be between0.05 and 0.001 g/cm³.

We claim:
 1. A liposome vesicle precursor in the form of a dry lipiddeposit, consisting of phospholipids selected from phosphatidic acid,phosphatidyl choline, phosphatidylethanol amine, phosphatidyl serine,phosphatidyl glycerol, phosphatidyl inositol and mixtures thereofwherein the lipid deposit is a three dimensional expanded foamedstructure with bulk density of below 0.1 g/cm³.
 2. A liposome vesicleprecursor in the form of a dry lipid deposit, selected from phosphatidicacid, phosphatidyl choline, phosphatidylethanol amine, phosphatidylserine, phosphatidyl glycerol, phosphatidyl inositol and mixturesthereof and also containing a substance selected from dicetylphosphate,cholesterol, ergosterol, phytosterol, sitosterol, lanosterol,(α-tocopherol, stearic acid, stearyl amine and mixtures thereof whereinthe lipid deposit is a three dimensional expanded foamed structure withbulk density of below 0.1 g/cm³.
 3. The vesicle precursor of claim 1 or2, wherein the bulk density of the expanded three dimensional lipiddeposit is below 0.08 g/cm³.
 4. The vesicle precursor of claim 3,wherein the bulk density of the expanded three dimensional lipid depositis between 0.05 and 0.001.
 5. The vesicle precursor of claim 4, whereinthe bulk density of the expanded three dimensional lipid deposit isbetween 0.02 and 0.01.
 6. The vesicle precursor of claim 2 or 3, whereinthe expanded three dimensional structure is supported by a network ofinert porous material.
 7. The vesicle precursor of claim 6, wherein theinert porous material comprises an array of tubing or an array of inertpacking.
 8. The vesicle precursor of claim 7, wherein the tubing has aninner diameter between 0.5 mm and 5 mm and wall thickness between 0.5and 2 mm.
 9. The vesicle precursor of claim 7, wherein the inert packingis selected from the group consisting of hollow glass spheres,reticulated carbon, reticulated vitreous carbon, reticulated metal,glass wool, metal wool, glass fiber and metal fiber.